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Updated: Jul 19, 2025
A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9
Published on: August 28, 2018
MiR-15b-5p and PCSK9 inhibition reduces lipopolysaccharide-induced endothelial dysfunction by targeting SIRT4
Elisa Martino1, Nunzia D'Onofrio2, Anna Balestrieri3
1Department of Precision Medicine, University of Campania Luigi Vanvitelli, Via L. De Crecchio 7, 80138, Naples, Italy.
Background:
Endothelial dysfunction and deregulated microRNAs (miRNAs) participate in the development of sepsis and are associated with septic organ failure and death. Here, we explored the role of miR-15b-5p on inflammatory pathways in lipopolysaccharide (LPS)-treated human endothelial cells, HUVEC and TeloHAEC.
Methods:
The miR-15b-5p levels were evaluated in LPS-stimulated HUVEC and TeloHAEC cells by quantitative real-time PCR (qRT-PCR). Functional experiments using cell counting kit-8 (CCK-8), transfection with antagomir, and enzyme-linked immunosorbent assays (ELISA) were conducted, along with investigation of pyroptosis, apoptosis, autophagy, and mitochondrial reactive oxygen species (ROS) by cytofluorometric analysis and verified by fluorescence microscopy. Sirtuin 4 (SIRT4) levels were detected by ELISA and immunoblotting, while proprotein convertase subtilisin-kexin type 9 (PCSK9) expression was determined by flow cytometry (FACS) and immunofluorescence analyses. Dual-luciferase reporter evaluation was performed to confirm the miR-15b-5p-SIRT4 interaction.
Results:
The results showed a correlation among miR-15b-5p, PCSK9, and SIRT4 levels in septic HUVEC and TeloHAEC. Inhibition of miR-15b-5p upregulated SIRT4 content, alleviated sepsis-related inflammatory pathways, attenuated mitochondrial stress, and prevented apoptosis, pyroptosis, and autophagic mechanisms. Finally, a PCSK9 inhibitor (i-PCSK9) was used to analyze the involvement of PCSK9 in septic endothelial injury. i-PCSK9 treatment increased SIRT4 protein levels, opposed the septic inflammatory cascade leading to pyroptosis and autophagy, and strengthened the protective role of miR-15b-5p inhibition. Increased luciferase signal validated the miR-15b-5p-SIRT4 binding.
Conclusions:
Our in vitro findings suggested the miR-15b-5p-SIRT4 axis as a suitable target for LPS-induced inflammatory pathways occurring in sepsis, and provide additional knowledge on the beneficial effect of i-PCSK9 in preventing vascular damage by targeting SIRT4.
Insights
Targeting the miR-15b-5p-SIRT4 axis in endothelial cells can mitigate sepsis-induced inflammation and vascular damage. Inhibition of miR-15b-5p and PCSK9 (proprotein convertase subtilisin-kexin type 9) shows protective effects against sepsis.
Area of Science:
- Molecular Biology
- Cell Biology
- Sepsis Research
Background:
- Endothelial dysfunction and microRNAs (miRNAs) are implicated in sepsis development and outcomes.
- Dysregulated miRNAs contribute to septic organ failure and mortality.
Purpose of the Study:
- To investigate the role of miR-15b-5p in lipopolysaccharide (LPS)-induced inflammatory pathways in human endothelial cells (HUVEC and TeloHAEC).
- To explore the therapeutic potential of targeting the miR-15b-5p-SIRT4 axis and PCSK9 in sepsis-related endothelial injury.
Main Methods:
- Quantitative real-time PCR (qRT-PCR) to measure miR-15b-5p levels.
- Cell counting kit-8 (CCK-8), antagomir transfection, and ELISA for functional assays.
- Flow cytometry and fluorescence microscopy to assess apoptosis, pyroptosis, autophagy, and reactive oxygen species (ROS).
- ELISA, immunoblotting, and FACS to detect SIRT4 and PCSK9 (proprotein convertase subtilisin-kexin type 9) expression.
- Dual-luciferase reporter assays to confirm miR-15b-5p and SIRT4 interaction.
Main Results:
- miR-15b-5p, PCSK9, and SIRT4 levels were correlated in septic endothelial cells.
- Inhibiting miR-15b-5p upregulated SIRT4, reduced inflammation, attenuated mitochondrial stress, and prevented cell death pathways (apoptosis, pyroptosis, autophagy).
- PCSK9 inhibition (i-PCSK9) increased SIRT4, counteracted sepsis-induced inflammation, pyroptosis, and autophagy, supporting miR-15b-5p inhibition's protective role.
Conclusions:
- The miR-15b-5p-SIRT4 axis is a potential therapeutic target for LPS-induced inflammatory pathways in sepsis.
- PCSK9 inhibition offers a protective effect against vascular damage in sepsis by targeting SIRT4.
